Expression of VCAM-1
Adhesion of leukocytes to the endothelium represents a fundamental, early event in a wide variety of inflammatory conditions, including atherosclerosis, autoimmune disorders and bacterial and viral infections. Leukocyte recruitment to the endothelium is started when inducible adhesion molecule receptors on the surface of endothelial cells interact with counterreceptors on immune cells. Vascular endothelial cells determine which type of leukocytes (monocytes, lymphocytes, or neutrophils) are recruited, by selectively expressing specific adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin. In the earliest stage of the atherosclerotic lesion, there is a localized endothelial expression of VCAM-1 and selective recruitment of mononuclear leukocytes that express the integrin counterreceptor VLA-4. Because of the selective expression of VLA-4 on monocytes and lymphocytes, but not neutrophils, VCAM-1 is important in mediating the selective adhesion of mononuclear leukocytes. Subsequent conversion of leukocytes to foamy macrophages results in the synthesis of a wide variety of inflammatory cytokines, growth factors, and chemoattractants that help propagate the leukocyte and platelet recruitment, smooth muscle cell proliferation, endothelial cell activation, and extracellular matrix synthesis characteristic of maturing atherosclerotic plaque.
VCAM-1 is also a mediator of chronic inflammatory disorders such as asthma, rheumatoid arthritis, autoimmune diabetes and multiple sclerosis. For example, it is known that the expression of VCAM-1 and ICAM-1 are increased in asthmatics (Pilewski, J. M., et al. Am. J. Respir. Cell Mol. Biol. 12, 1-3 (1995); Ohkawara, Y., et al., Am. J. Respir. Cell Mol. Biol. 12, 4-12 (1995)). Additionally, blocking the integrin receptors for VCAM-1 and ICAM-1 (VLA-4 and LFA-1, respectively) suppressed both early and late phase responses in an ovalbumin-sensitized rat model of allergic airway responses (Rabb, H. A., et al., Am. J. Respir. Care Med. 149, 1186-1191 (1994)). There is also increased expression of endothelial adhesion molecules, including VCAM-1, in the microvasculature of rheumatoid synovium (Koch, A. E. et al., Lab. Invest. 64, 313-322 (1991); Morales-Ducret, J. et al., Immunol. 149, 1421-1431 (1992)). Neutralizing antibodies directed against VCAM-1 or its counter receptor, VLA-4, can delay the onset of diabetes in a mouse model (NOD mice) which spontaneously develop the disease (Yang, X. D. et al., Proc. Natl. Acad. Sci. U.S.A. 90, 10494-10498 (1993); Burkly, L. C. et al., Diabetes 43, 523-534 (1994); Baron, J. L. et al., J. Clin. Invest. 93, 1700-1708 (1994)).
VCAM-1 is expressed by cells both as a membrane bound form and as a soluble form. The soluble form of VCAM-1 has been shown to induce chemotaxis of vascular endothelial cells in vitro and stimulate an angiogenic response in rat cornea (Koch, A. F. et al., Nature 376, 517-519 (1995)). Inhibitors of the expression of soluble VCAM-1 have potential therapeutic value in treating diseases with a strong angiogenic component, including tumor growth and metastasis (Folkman, J., and Shing, Y., Biol Chem. 10931-10934 (1992)).
VCAM-1 is expressed in cultured human vascular endothelial cells after activation by lipopolysaccharide (LPS) and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha). These factors are not selective for activation of cell adhesion molecule expression.
It has been documented that VCAM-1 is expressed on brain microvessel endothelial cells in active lesions of multiple sclerosis brain. Multiple sclerosis is a common demyelinating disorder for the central nervous system, causing patches of sclerosis (plaques) in the brain and spinal cord. It occurs in young adults and has protean clinical manifestations. Experimental therapy using antibodies for VCAM-1 in autoimmune encephalomyelitis, which is an animal model for multiple sclerosis, has shown that adhesion molecules play a role in the pathogenesis of the disease (Benveniste et al., J. Neuroimmunol. 98:77-88, 1999). Time and dose dependent expression of VCAM-1 and release of soluble VCAM-1 were detected in cultures of human cerebral endothelial cells induced by TNF-alpha, but not in peripheral blood mononuclear cells (Kallmann et al., Brain 123:687-697, 2000). Clinical data also show that adhesion molecules in blood and cerebrospinal fluid are up-regulated throughout the clinical spectrum of multiple sclerosis, further supporting the belief that multiple sclerosis can be suppressed by interfering with cell adhesion molecules such as VCAM-1 (Elovaara et al., Arch. Neurol. 57:546-551, 2000).
Cell adhesion molecules have also been shown to play an important role in recruiting T lymphocytes to graft endothelium on transplanted vascularized tissue and organs, and have thus been implicated in organ transplant rejections and allograft rejections. Expression on VCAM-1 and ICAM-1 in normal cardiac tissue is very limited in vascular endothelium. However, immunohistochemical studies on transplanted allografts have revealed that increased expression of these molecules on the vascular endothelium and on cardiac myocytes occurs from the early stages of graft rejection in human and animal transplants (Cosimi et al., J. Immunol. 144: 4604-4612, 1990; Orosz et al., Transplantation 56:453-460, 1993; Pelletier et al., Transplant Proc. 25: 839-841, 1993; Tanio et al. Circulation 89:1760-1768, 1994). In a murine heart transplant model, the survival rate of mice treated with anti-VCAM-1 monoclonal antibodies or anti-VLA-4 monoclonal antibodies was greater than the survival rate of a control group (Isobe et al., J. Immunol. 153: 5810-5818, 1994; Orosz et al., J. Heart Lung Transplant 16: 889-904, 1997).
Hypercholesterolemia and Hyperlipidemia
Hypercholesterolemia is an important risk factor associated with cardiovascular disease. Serum lipoproteins are the carriers for lipids in the circulation. Lipoproteins are classified according to their density: chylomicrons, very low-density lipoproteins (VLDL), low density lipoproteins (LDL) and high-density lipoproteins (HDL). Chylomicrons primarily participate in transporting dietary triglycerides and cholesterol from the intestine to adipose tissue and liver. VLDL deliver endogenously synthesized triglycerides from liver to adipose and other tissues. LDL transports cholesterol to peripheral tissues and regulates endogenous cholesterol levels in those tissues. HDL transports cholesterol from peripheral tissues to the liver. Arterial wall cholesterol is derived almost exclusively from LDL. Brown and Goldstein, Ann. Rev. Biochem. 52, 223 (1983); Miller, Ann. Rev. Med. 31, 97 (1980).
Steinberg, et al., (N. Eng. J. Med. 1989; 320:915-924) hypothesized that modification of low-density lipoprotein (LDL) into oxidatively modified LDL (ox-LDL) by reactive oxygen species is the central event that initiates and propagates atherosclerosis. Oxidized LDL is a complex structure consisting of at least several chemically distinct oxidized materials, each of which, alone or in combination, may modulate cytokine-activated adhesion molecule gene expression. Fatty acid hydroperoxides such as linoleyl hydroperoxide (13-HPODE) are produced from free fatty acids by lipoxygenases and are an important component of oxidized LDL.
Through a mechanism that is not well defined, areas of vessel wall predisposed to atherosclerosis preferentially sequester circulating LDL. Through a poorly understood pathway, endothelial, smooth muscle, and/or inflammatory cells then convert LDL to ox-LDL. In contrast to LDL, which is taken up through the LDL receptor, monocytes avidly take up ox-LDL through a “scavenger” receptor whose expression, unlike the LDL receptor, is not inhibited as the content of intracellular lipid rises. Thus, monocytes continue to take up ox-LDL and become lipid-engorged macrophage-foam cells that form the fatty streak.
Typically, cholesterol is carried in the blood of warm-blooded animals in certain lipid-protein complexes such as chylomicrons, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). It is widely accepted that LDL functions in a way that directly results in deposition of the LDL cholesterol in the blood-vessel wall and that HDL functions in way that results in the HDL picking up cholesterol from the vessel wall and transporting it to the liver where it is metabolized (Brown and Goldstein, Ann. Rev. Biochem. 52, 223 (1983); Miller, Ann. Rev. Med. 31, 97 (1980)). For example, in various epidemiologic studies the LDL cholesterol levels correlate well with the risk of coronary heart disease whereas the HDL cholesterol levels are inversely associated with coronary heart disease (Patton et al., Clin. Chem. 29, 1980 (1983)). In patients with low levels of LDL, the development of atherosclerosis is rare.
Furthermore, there is evidence based on animal and laboratory findings that peroxidation of LDL lipid, such as the unsaturated fatty acid portions of LDL cholesteryl esters and phospholipids, facilitate the accumulation of cholesterol in monocyte/macrophages which eventually are transformed into foam cells and become deposited in the sub-endothelial space of the vessel wall. The accumulation of foam cells in the vessel wall is recognized as an early event in the formation of an atherosclerotic plaque. Thus it is believed that peroxidation of LDL lipid is an important prerequisite to the facilitated accumulation of cholesterol in the vessel wall and the subsequent formation of an atherosclerotic plaque. (Parthasarathy et al., J. Clin. Invest. 77,641 (1986)). It is therefore desirable to provide methods of inhibiting LDL lipid peroxidation in a patient in need thereof.
Elevated cholesterol levels are associated with a number of disease states, including restenosis, angina, cerebral atherosclerosis, and xanthoma. It is desirable to provide a method for reducing plasma cholesterol in patients with, or at risk of developing, restenosis, angina, cerebral arteriosclerosis, xanthoma, and other disease states associated with elevated cholesterol levels.
There are several drug classes that are commonly used to lower LDL levels, including bile acid sequestrants, nicotinic acid (niacin), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins). Statins are among the most effective agents currently on the market for hypercholesterolemia, and include pravastatin (Pravchol, Bristol Myers Squibb), atorvastatin (Warner Lambert/Pfizer), simvastatin (Zocor, Merck), lovastatin (Mevacor, Merck), and fluvastatin (Lescol).
Probucol has been shown to possess potent antioxidant properties and to block oxidative modification of LDL. Consistent with these findings, probucol has been shown to actually slow the progression of atherosclerosis in LDL receptor-deficient rabbits as discussed in Carew et al. Proc. Natl. Acad. Sci. U.S.A. 84:7725-7729 (1987).
Probucol is chemically related to the widely used food additives 2, (3)-tert-butyl-4-hydroxyanisole (BHA) and 2,6-di-tert-butyl-4-methyl phenol (BHT). It is a thioketal having a chemical name of 4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol) and has the following chemical structure: 
Probucol is used primarily to lower serum cholesterol levels in hypercholesterolemic patients. Probucol is commonly administered in the form of tablets available under the trademark Lorelco™.
U.S. Pat. No. 5,262,439 to Parthasarathy discloses analogs of probucol with increased water solubility in which one or both of the hydroxyl groups are replaced with ester groups.
A series of French patents disclose that certain probucol ester derivatives are hypocholesterolemic and hypolipemic agents: Fr 2168137 (bis 4-hydroxyphenylthioalkane esters); Fr 2140771 (tetralinyl phenoxy alkanoic esters of probucol); Fr 2140769 (benzofuryloxyalkanoic acid derivatives of probucol); Fr 2134810 (bis-(3-alkyl-5-t-alkyl-4-thiazole-5-carboxy)phenylthio)alkanes; FR 2133024 (bis-(4-nicotinoyloxyphenylthio)propanes; and Fr 2130975 (bis(4-(phenoxyalkanoyloxy)-phenylthio)alkanes).
De Meglio et al., have described several ethers of symmetrical molecules for the treatment of hyperlipidemia. These molecules contain two phenyl rings attached to each other through a —S—C(CH3)2—S— bridge. In contrast to probucol, the phenyl groups do not have t-butyl as substituents. (De Meglio et al., New Derivatives of Clofibrate and probucol: Preliminary Studies of Hypolipemic Activity; Farmaco, Ed. Sci (1985), 40 (11), 833-44).
WO 00/26184 disclosed a large genus of compounds with a general formula of phenyl-S-alkylene-S-phenyl, in which one or both phenyl rings can be substituted at any position. These compounds were disclosed as lubricants.
U.S. Pat. Nos. 5,750,351; 5,807,884; 5,811,449; 5,846,959; 5,773,231, and 5,773,209 to Medford, et al. (assigned to Emory University), as well as the corresponding WO95/30415 to Emory University indicate that polyunsaturated fatty acids (“PUFAs”) and their hydroperoxides (“ox-PUFAs”), which are important components of oxidatively modified low density lipoprotein (LDL), induce the expression of VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1) or E-selectin in human aortic endothelial cells, through a mechanism that is not mediated by cytokines or other noncytokine signals.
U.S. Pat. No. 5,155,250 to Parker, et al. discloses that 2,6-dialkyl-4-silylphenols are antiatherosclerotic agents. The same compounds are disclosed as serum cholesterol lowering agents in PCT Publication No. WO 95/15760, published on Jun. 15, 1995. U.S. Pat. No. 5,608,095 to Parker, et al. discloses that alkylated-4-silyl-phenols inhibit the peroxidation of LDL, lower plasma cholesterol, and inhibit the expression of VCAM-1, and thus are useful in the treatment of atherosclerosis.
A series of European patent applications of Shionogi Seiyaku Kabushiki Kaisha disclose phenolic thioethers for use in treating arteriosclerosis. European Patent Application No. 348 203 discloses phenolic thioethers which inhibit the denaturation of LDL and the incorporation of LDL by macrophages. The compounds are useful as anti-arteriosclerosis agents. Hydroxamic acid derivatives of these compounds are disclosed in European Patent Application No. 405 788 and are useful for the treatment of arteriosclerosis, ulcer, inflammation and allergy. Carbamoyl and cyano derivatives of the phenolic thioethers are disclosed in U.S. Pat. No. 4,954,514 to Kita, et al.
U.S. Pat. No. 4,752,616 to Hall, et al., discloses arylthioalkylphenylcarboxylic acids for the treatment of thrombotic disease. The compounds disclosed are useful as platelet aggregation inhibitors for the treatment of coronary or cerebral thromboses and the inhibition of bronchoconstriction, among others.
A series of patents to Adir et Compagnie disclose substituted phenoxyisobutyric acids and esters useful as antioxidants and hypolipaemic agents. This series includes U.S. Pat. Nos. 5,206,247 and 5,627,205 to Regnier, et al. (which corresponds to European Patent Application No. 621 255) and European Patent Application No. 763 527.
WO 97/15546 to Nippon Shinyaku Co. Ltd. discloses carboxylic acid derivatives for the treatment of arterial sclerosis, ischemic heart diseases, cerebral infarction and post PTCA restenosis.
The Dow Chemical Company is the assignee of patents to hypolipidemic 2-(3,5-di-tert-butyl-4-hydroxyphenyl)thio carboxamides. For example, U.S. Pat. Nos. 4,029,812, 4,076,841 and 4,078,084 to Wagner, et al., disclose these compounds for reducing blood serum lipids, especially cholesterol and triglyceride levels.
PCT WO 98/51289, filed by Emory University and listing as inventors Russell M. Medford and Patricia K. Somers, claims priority to provisional patent application U.S. Ser. No. 60/047,020, filed on May 14, 1997. This application discloses that monoesters of probucol inhibit the expression of VCAM-1, and may also exhibit the composite profile of lowering LDL and reducing cholesterol.
Given that cardiovascular disease is currently the leading cause of death in the United States, and ninety percent of cardiovascular disease is presently diagnosed as atherosclerosis, there is a strong need to identify new methods and pharmaceutical agents for its treatment. Moreover, given the growing body of evidence implicating inflammation and VCAM-1 expression in cardiovascular disease and other disease states such as rheumatoid arthritis, multiple sclerosis, and allograft rejection, there is also a need to identify methods and pharmaceutical agents for reducing inflammation and VCAM-1 expression.
It is therefore an object of the invention to provide methods of treating disorders and diseases mediated by VCAM-1, including cardiovascular and inflammatory diseases.
It is also an object to provide new classes of compounds and pharmaceutical compositions for the treatment of diseases and disorders mediated by the expression of VCAM-1.
It is a further object of the present invention to provide methods, compounds, and compositions for the treatment of hypercholesterolemia and/or hyperlipidemia.
It is also an object of the present invention to provide new classes of thioketal and thioether compounds, and pharmaceutical uses of such compounds.